Nucleic acid amplification technology (NAAT) is an invaluable and powerful tool in many areas of research and diagnosis. Such NAAT techniques allow detection and quantitation of a nucleic acid in a sample with high sensitivity and specificity as well as quantitative analysis of nucleic acids in a sample.
Nucleic acid amplification may be used to determine the presence of a particular template nucleic acid in a sample, as indicated by the presence of an amplification product following the implementation of a particular NAAT. Conversely, the absence of any amplification product indicates the absence of template nucleic acid in the sample. Such techniques are of great importance in diagnostic applications, for example, for determining whether a pathogen is present in a sample.
The prior art has described a variety of thermocycling and isothermal techniques for amplification of nucleic acids. Thermocycling techniques, such as the polymerase chain reaction (PCR), use temperature cycling to drive repeated cycles of DNA synthesis leading to large amounts of new DNA being synthesised in proportion to the original amount of template DNA. A number of isothermal techniques have also been developed that do not rely on thermocycling to drive the amplification reaction. Isothermal techniques, which utilise DNA polymerases with strand-displacement activity, have been developed for amplification reactions that do not involve an RNA-synthesis step. Similarly, for amplification reactions that do involve an RNA-synthesis step, isothermal techniques have been developed that may use reverse transcriptase, RNase H and/or a DNA-dependent RNA polymerase (see for example, Nucleic Acid Isothermal Amplification Technologies—A Review. Nucleosides, Nucleotides and Nucleic Acids, Volume 27, Issue 3 Mar. 2008, pages 224-243).
One thing all NAAT techniques have in common is that it is often essential that the reaction is monitored by suitable controls in order to ensure that a negative result is actually due to the absence of the nucleic acid rather than due to other factors, for example the presence of inhibitors in a sample. The prior art describes several methods to achieve this.
One method is to perform two amplification reactions in separate vessels in parallel. One vessel contains the test sample and the other contains a nucleic acid of a known sequence, which serves as a positive control, in addition to the test sample. If no amplification is detected in the test sample but an amplification product can be detected in the control sample, the test can be considered true negative. Likewise, if no amplification product is present in the control, an inhibitor must be present in the sample.
The use of an internal control, i.e. a control which is amplified in the same reaction vessel, has some advantages over using two vessels to establish whether inhibitors are present. Firstly, fewer tubes and reagents are required, thereby reducing the unit cost per test where an inhibitor control is absolutely required. Secondly, fewer manipulations are required. Thirdly, since fewer tubes are required, more samples can be analysed per unit capacity of the hardware used to run the reaction. For example, using a standard 96 well detection system, 96 samples can be analysed using an internal control whereas only 48 samples can be analysed where two vessels must be used per test.
The internal control method presents the technical challenge of differentiating between the signal resulting from the target polynucleotide in the sample and the signal resulting from the control nucleic acid. To detect the signals from a sample and the control nucleic acid in the same vessel, it is necessary that the sequence of the control nucleic acid has some associated difference from the target nucleic acid to allow a detection system to differentiate between the two amplified products. Further still, there needs to be some means to differentiate the respective signals from the two amplification processes. This has been achieved by the use of separate reporter systems for the test-sample and control respectively. For example, two fluorescent probes may be employed of different emission maxima (or different enough for their respective signals to be differentiated) one which only gives a signal on binding to the products of the test-sample amplification process, and one which only gives a signal when binding to the products of the control products. In this way, by detecting two independent signals from a sample, it is possible to follow the respective amplification processes in the same reaction vessel.
Alternatively, where appropriate, a melt-curve analysis of the results of an amplification reaction can be performed to assess if there is signal from the test-sample and control. This may or may not encompass the use of fluorescent probes (see, for example EP1109934). The test-sample and the control are thereby amplified with the same reaction kinetics. The disadvantage of the melt-curve analysis is that it requires an additional step after the amplification reaction in order to detect the control which does not only elongate the process but also adds significant complexity to the hardware which is required to detect the control and the amplified polynucleic acid.
Where two or more reporter systems must be employed, the hardware used to detect the reporter must be sophisticated enough to perform measurements and differentiate at least two reporter signals. Further, for practical applications in diagnostics, these readings must be performed on a small sample, or multiple small samples: typically reactions volumes are between 10-100 μl to avoid high reagent costs per test. This requires very sophisticated hardware which is very expensive.
Hence, there is a need in the art for improved detection methods for detection of nucleic acids and internal controls in the same reaction vessel. In particular, a method whereby an internal control could be monitored without the need for two separate reporter signals to be independently measured, would be of great benefit as this would omit the necessity for expensive hardware that can measure multiple signals following NAAT. Furthermore there are some NAAT techniques which utilize reporter technologies whereby it is not possible to measure more than one type of signal from a sample. Such methods include reporter systems based on bioluminescence (published International patent applications WO 2004/062338 & WO 2006/010948), turbidity (published International patent application WO 01/83817) or certain electrochemical methodologies.